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MitoPedia alert - 2020
- Answered Questions from the »Oroboros Ecosystem Agenda 2020 Archive«
Dec 2020
 
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Do you know which graph layout to choose in DatLab?
Different layouts are required depending on which instrumental quality control or measurement with biological sample is performed. Learn more about Standard layouts provided in DatLab:
- »Layout for DatLab graphs«
- communicated by »Schmitt Sabine« and »Komlodi Timea«
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Can data be compared if different gain settings were used?
Yes, oxygen signals obtained after proper calibration are independent of the gain setting, and can therefore be grouped together for comparison and analysis.
- »Gain«
- communicated by »Cecatto Cristiane« and »Schmitt Sabine«
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Nov 2020
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Cell culture medium and dithionite titrations – a bad combination for an instrumental O2 background test?
Cell culture media are not ideal for instrumental O2 background test when using dithionite titrations because side reactions affecting the O2 concentration and thus interfering with the test. Instead of cell culture media, use MiR05, MiR06 or a strongly buffered alkaline phosphate solution.
-»MiPNet14.06 Instrumental O2 background«
- communicated by »Komlodi Timea« and »Cecatto Cristiane«
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Have you observed a high signal at zero oxygen?
A high signal at zero oxygen might be due to a technical issue with the O2k-Main Unit, the OroboPOS-Connector, the OroboPOS or a bad electrical connection between these components. The high zero current test helps you to locate the cause.
- »High signal at zero oxygen«
- communicated by »Schmitt Sabine« and »Komlodi Timea«
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Oct 2020
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Did you know that oxygen flux can be converted to flux control ratios (FCRs) directly in DatLab?
Use this function to easily plot your experimental traces as FCRs, or to analyze your data.
- »FCR in DatLab«
- communicated by »Komlodi Timea« and »Cecatto Cristiane«
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Did you know that safranin can exert an inhibitory effect on mitochondrial respiration?
Application of safranin leads to partial inhibition of NADH-linked respiration and above 2 µM on the succinate-linked respiration. By running a control high-resolution respirometry experiment without the fluorophore, you can detect and do quality control for this inhibitory effect.
- You can read more about it in the following publication: »Krumschnabel 2014 Methods Enzymol«
- communicated by »Komlodi Timea« and »Cecatto Cristiane«
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If using the Amplex UltraRed assay, when should the background H2O2 calibration be performed?
Background H2O2 calibration needs to be performed for each individual instrumental setting (O2k, fluorescence sensor and chamber), if a new stock solution of DTPA, HRP, SOD, Amplex UltraRed, or H2O2 is prepared, or if a different respiration medium is used. As the H2O2 stock solution needs to be freshly prepared, it is recommended to perform a background H2O2 calibration before each experiment and at a minimum once per experimental day.
- »How to find the background H2O2 calibration protocol in DatLab « select “AmR calibration” for the background H2O2 calibration protocol
- communicated by »Komlodi Timea« and »Schmitt Sabine«
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Sep 2020
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Are whitish deposits forming on the glass wall of the O2k-Chamber?
These deposits might be an accumulation of proteins and subsequent embedding of other biological material or small glass splinters, and a sign that the O2k-Chamber requires cleaning.
For further details on how to clean the O2k-Chamber properly, see:
- »Cleaning the O2k-chamber«
- communicated by »Komlodi Timea«, »Sabine Schmitt«, and »Lisa Tindle-Solomon«
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Did you know the reverse electron flow from Complex II to Complex I can be investigated in mitochondrial preparations by measuring hydrogen peroxide production using Amplex UltraRed assay?
The reverse electron flow, also called reverse electron transfer (RET), happens when electrons are transferred from Complex II to the Q-junction and then back to Complex I at high mt-membrane potential. This phenomenon results in high hydrogen peroxide production in some mitochondrial preparations. The SUIT-026 protocol was designed to investigate this effect.
- »SUIT-026«
- communicated by »Komlodi Timea« and »Cardoso Luiza«
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Why do we perform multiple H2O2 calibrations in the Amplex® UltraRed assay?
The sensitivity to H2O2 of the fluorometric signal changes during the experiment as a function of: (1) experimental time and accumulating UltroxRed (similar to resorufin), (2) changes of the optical properties due to titrations, and (3) the radical scavenging capacity of the sample. Therefore, H2O2 calibrations are performed before and after sample addition, and after selected titration steps.
- »Komlodi 2018 Methods Mol Biol«
The DLPs for AmR SUIT protocols already come with multiple steps of H2O2 calibrations implemented.
- » SUITbrowser«
- communicated by »Komlodi Timea« and »Cardoso Luiza«
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Aug 2020
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What do you do if values, symbols, and/or units are not correctly displayed in the DatLab software?
It is possible to retrieve the values, symbols and/or correct units by updating the Windows control panel settings.
- » Troubleshooting for problems with text in DatLab«
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Jul 2020
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How can you compare respirometric experiments using an internal normalization?
Flux control ratios (FCR) are independent of external measurements (e.g. protein determination, citrate synthase activity) and therefore can be used to analyze data when other normalization methods cannot be applied. Learn more about how to use this option to assess qualitative changes of mitochondrial respiratory control with DatLab and the “O2 analysis template DL7.4”.
- »Flux control ratio«
Read more: Gnaiger Erich et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1 »Bioblast link«
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Did you know that you can apply instrumental O2 background test parameters (i.e., intercept and slope) from an existing file in DatLab?
In the DatLab file, you can copy the Instrumental O2 background test parameters directly via the ‘Copy from file’ option in the ‘Slope configuration’ window.
For more information, see:
- »Copy from file«
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Jun 2020
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Did you know that instabilities of the slope of the oxygen signal (O2 slope neg.) can be prevented by the proper use of TIP2k-Filter Papers during an instrumental O2 background test?
- »Unstable 'O2 slope neg.'« and »TIP2k Filter Papers«
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Mitochondria show variable preferences for glutamate & malate or pyruvate & malate to support NADH-linked respiration, depending on species, tissues, and pathophysiological state.
DatLab 7.4 provides a step-by-step guide through SUIT-008 and SUIT-014, which can be used in parallel to compare respiratory capacities supported by these NADH-linked fuel substrates.
-»SUIT-008« and »SUIT-014«
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Did you know that experimental artefacts can occur in the Amplex UltraRed assay after injection of chemicals that interfere with the fluorescence signal?
- » Injection artefact«
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Is the noise seen in the slope of the oxygen signal (O2 slope neg.) within recommended values?
- An 'O2 slope neg.' within ± 2 pmol∙s−1∙mL−1 is optimal (± 4 pmol∙s−1∙mL−1 is acceptable) at a data recording interval of 2 s and 40 data points selected for calculation of the slope.
- » Air and zero calibration«
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May 2020
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Do you know how the "switch components" approach can help you find what is causing your O2k to not reach the quality control specifications?
Do not despair if specifications given in the O2k-Manuals are not obtained. See how to troubleshoot on your own.
- »Switch components approach«
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Do you know that a humid O2k-environment and/or liquid spillage may lead to a high raw POS signal [V]?
See how to clean and dry the electrical connections of the OroboPOS-Connector.
- »Cleaning the electrical connections«
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Do you know how to avoid underestimating/overestimating respiratory fluxes? Measuring flux at steady states with high-resolution respirometry is the key to obtaining correct values.
Learn more about steady states including performance and mark setting for data analysis.
- »Steady state«
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Apr 2020
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Do you set marks correctly in the instrumental O2 background test when planning to apply the background correction to a SUIT protocol with a high O2 regime?
The range of O2 concentrations selected in the instrumental background test should correspond to the range of experimental O2 concentrations.
- » O2 Background correction - troubleshooting«
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Did you know that some substrates alone cannot maintain a stable mitochondrial respiratory flux in OXPHOS?
Unstable respiratory fluxes in OXPHOS might be due to substrates limitation.
- » Pyruvate - troubleshooting«
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Why are the oxygen levels different between chambers prior to my experimental run?
After switching on the O2k, air calibration has to be performed; otherwise the displayed concentrations may deviate from accurate concentrations.
- » Oxygen calibration - troubleshooting«
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Did you know that you can edit the background parameters in the 'O2 analysis template DL7.4?
If you did not apply a finally evaluated O2 background correction to your experimental DatLab file, you can edit the O2 background parameters directly in the O2 analysis template DL7.4 for completion of Oxygen flux analysis - DatLab 7.4.
- »SUIT: Browse DL-Protocols and templates«
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Do you want to search the best SUIT protocol for your research questions?
- Use the SUITbrowser: »SUITbrowser«
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Mar 2020
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Do you know that you can use the Titration-Injection microPump (TIP2k) to perform an instrumental O2 background test when using the O2k-sV-Module?
(1) By adjusting the dithionite concentration to 2.5 mM, the instrumental O2 background test can be performed with the "BG Feedback" settings. - »Dithionite«
(2) By adjusting the TIP2k setup with specific settings for the O2k-sV-Module, the instrumental O2 background test can be performed using the O2k-sV-Module. - »O2k-sV-Module Manual«
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Do you change the slope smoothing to 20 while running experiments with biological sample?
While running your experiment, especially during uncoupler titrations, it is important to decrease the slope smoothing in order to have a faster visualization of the titrations effect on respiration.
- »Slope smoothing«
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Feb 2020
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Calibration parameters are transferred to experimental DatLab files
Save calibration information from selected Marks while DatLab is connected to the O2k during an instrumental calibration run. Air calibration and zero oxygen calibration can be performed and saved in a single DatLab file or in separate DatLab files.
- » Oxygen calibration - DatLab«
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Jan 2020
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Reproducibility committment in high-resolution respirometry: quality control in oxygen calibration
In the DatLab 7.4 software, new instrumental DL-Protocols guide the user automatically step-by-step, to calibrate the instrument accurately. Reproducibility is monitored and documented when following user-friendly standard operating procedures (SOP).
- »Oxygen calibration - DatLab«
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Do you check the O2 flux at the end of the air calibration with the O2k-chamber closed?
In DatLab 7.4, instrumental DL-Protocols guide you automatically towards closing the O2k-chamber after air calibration, to monitor "O2 slope neg." (negative) as a test of any biological contamination which would increase oxygen consumption in the absence of a biological sample.
- »Oxygen calibration - DatLab«
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Do you set mark ‘R1’ and complete the air calibration correctly?
Your air calibration is not effective until you set mark ‘R1’ and click on Calibrate and copy to clipboard in the O2 calibration window.
- »Oxygen calibration - DatLab«
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Do you write the serial number of the polarographic oxygen sensor (POS) in the ‘O2k configuration’ window?
Including the POS serial number in the ´O2k configuration´ window is important to keep a track record of the sensors used according to the O2k. This will be especially helpful if troubleshooting is needed.
- »O2k configuration«
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Do you perform O2k-chamber volume calibration?
The volume calibration is essential to ensure that the chamber volume is correctly calibrated to its standard volume of 2 mL (or 0.5 mL for the O2k-sV-Module).
- »O2k-chamber«
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Do you use the correct O2 solubility factor (FM) in the ‘O2 calibration’ window in DatLab?
The O2 solubility factor varies according to the medium used and influences the air calibration.
- »Oxygen solubility factor«
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Summary
Stay alert
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Test - Ecosystem agenda
